Identification of mycorrhiza-regulated genes with arbuscule development-related expression profile

Suppressive subtractive hybridisation was applied to the analysis of late stage arbuscular mycorrhizal development in pea. 96 cDNA clones were amplified and 81, which carried fragments more than 200 nt in size, were sequence analysed. Among 67 unique fragments, 10 showed no homology and 10 were similar to sequences with unknown function. RNA accumulation of the corresponding 67 genes was analysed by hybridisation of macro-arrays. The cDNAs used as probes were derived from roots of wild type and late mutant pea genotypes, inoculated or not with the AM fungus Glomus mosseae. After calibration, a more than 2.5-fold mycorrhiza-induced RNA accumulation was detected in two independent experiments in the wild type for 25 genes, 22 of which seemed to be induced specifically during late stage AM development. Differential expression for 7 genes was confirmed by RT-PCR using RNA from mycorrhiza and from controls of a different pea cultivar. In order to confirm arbuscule-related expression, the Medicago truncatula EST data base was screened for homologous sequences with putative mycorrhiza-related expression and among a number of sequences with significant similarities, a family of trypsin inhibitor genes could be identified. Mycorrhiza-induced RNA accumulation was verified for five members by real-time PCR and arbuscule-related activation of the promoter could be shown in transgenic roots for one of the genes, MtTi1.     

Growth promotion of Chinese cabbage and Arabidopsis by Piriformospora indica is not stimulated by mycelium-synthesized auxin

Piriformospora indica, an endophytic fungus of the order Sebacinales, interacts with the roots of a large variety of plant species. We compared the interaction of this fungus with Chinese cabbage (Brassica campestris subsp. chinensis) and Arabidopsis seedlings. The development of shoots and roots of Chinese cabbage seedlings was strongly promoted by P. indica and the fresh weight of the seedlings increased approximately twofold. The strong stimulation of root hair development resulted in a bushy root phenotype. The auxin level in the infected Chinese cabbage roots was twofold higher compared with the uncolonized controls. Three classes of auxin-related genes, which were upregulated by P. indica in Chinese cabbage roots, were isolated from a double-subtractive expressed sequence tag library: genes for proteins related to cell wall acidification, intercellular auxin transport carrier proteins such as AUX1, and auxin signal proteins. Overexpression of B. campestris BcAUX1 in Arabidopsis strongly promoted growth and biomass production of Arabidopsis seedlings and plants; the roots were highly branched but not bushy when compared with colonized Chinese cabbage roots. This suggests that BcAUX1 is a target of P. indica in Chinese cabbage. P. indica also promoted growth of Arabidopsis seedlings but the auxin levels were not higher and auxin genes were not upregulated, implying that auxin signaling is a more important target of P. indica in Chinese cabbage than in Arabidopsis. The fungus also stimulated growth of Arabidopsis aux1 and aux1/axr4 and rhd6 seedlings. Furthermore, a component in an exudate fraction from P. indica but not auxin stimulated growth of Chinese cabbage and Arabidopsis seedlings. We propose that activation of auxin biosynthesis and signaling in the roots might be the cause for the P. indica-mediated growth phenotype in Chinese cabbage.     

Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica

The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica.     

A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana

Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single-copy gene DMI-1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica-insensitive-2; pii-2), and a corresponding insertion line, are impaired in a leucine-rich repeat protein (At1g13230). The protein pii-2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X-100-insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine-rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii-2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica-induced growth promotion and enhanced seed production in Arabidopsis thaliana.     

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.     

Accumulation of alfalfa mosaic virus RNAs 1 and 2 requires the encoded proteins in cis.

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (A1MV) encode the replicase proteins P1 and P2, respectively. P1 expressed in transgenic plants (P1 plants) can be used in trans to support replication of A1MV RNAs 2 and 3, and P2 expressed in transgenic plants (P2 plants) can be used in trans to support replication of A1MV RNAs 1 and 3. Wild-type RNA 1 was able to coreplicate with RNAs 2 and 3 in P1 plants, but this ability was abolished by frameshifts or deletions in the P1 gene of RNA 1. Similarly, wild-type RNA 2 coreplicated with RNAs 1 and 3 in P2 plants, but frameshifts or deletions in the P2 gene of RNA 2 interfered with this replication. Apparently, the P1 and P2 genes are required in cis for the accumulation of RNAs 1 and 2, respectively. Point mutations in the GDD motif of the P2 gene in RNA 2 interfered with accumulation of RNA 2 in P2 plants, indicating that replication of RNA 2 is linked to its translation into a functional protein. Plants transformed with both the P1 and P2 genes (P12 plants) accumulate replicase activity that is able to replicate RNA 3 in trans. An analysis of the time course of the accumulation of RNAs 1, 2, and 3 in protoplasts of P12 plants supported the conclusion that translation and replication are tightly coupled for A1MV RNAs 1 and 2 but not for RNA 3.     

Piriformospora indica-a mutualistic basidiomycete with an exceptionally large plant host range

Piriformospora indica is a basidiomycete of the order Sebacinales, representing a model for the study of mutualistic symbiosis and, beyond that, the plant immune system. The fungus colonizes the roots of a wide range of vascular plants, increasing their growth, seed yield and adaptation to abiotic and biotic stresses. The fungal colonization of roots begins with a biotrophic growth phase, in which living cells are colonized, and continues with a cell death-dependent phase, in which root cells are actively killed by the fungus. The complexity of sebacinalean symbiosis is further enhanced by the presence of endocellular bacteria which may represent significant determinants for a successful outcome of the symbioses. Molecular ecological analyses have revealed an exceptional relevance of sebacinoid fungi in natural ecosystems worldwide. This natural competence could be rooted in their phenotypic adaptability, which, for instance, allows P. indica to grow readily on various synthetic media and to colonize distinct hosts. In molecular and genetic studies, P. indica's mutualistic colonization strategy has been partly unravelled, showing that the jasmonate pathway is exploited for immune suppression and successful development in roots. Research on P. indica supports efforts to make the bioprotective potential of the fungus accessible for agricultural plant production. The decoding of P. indica's genome has revealed its potential for application as bioagent and for targeted improvement of crop plants in biotechnology-based approaches.     

Comparative differential RNA display analysis of arbuscular mycorrhiza in Pisum sativum wild type and a mutant defective in late stage development

In order to analyse gene expression associated with the late stages of arbuscular mycorrhizal development between Pisum sativum and Glomus mosseae, comparative differential RNA display was carried out using wild-type P. sativum and a mutant, RisNod24, where the fungal partner is not able to form functional arbuscules. Comparison of RNA accumulation patterns between controls, G. mosseae-colonized mutant and wild-type roots resulted in the identification of four differentially occurring cDNA fragments. One of the corresponding genes was from the fungus and three of plant origin. One plant gene, Psam4 (P. sativum arbuscular mycorrhiza-regulated), was analysed in more detail. Sequencing of a cDNA clone showed that Psam4 encodes a proline-rich protein. Northern blot analysis and quantitative RT-PCR revealed a higher basal level of Psam4 RNA accumulation in the mutant compared to the wild type. In both pea genotypes, RNA accumulation was reduced after inoculation with mycorrhiza- or nodule-forming symbiotic microorganisms, but enhanced after infection with a root pathogenic fungus.     

The difference of cadmium accumulation between the indica and japonica subspecies and the mechanism of it

Many studies have shown genotypic differences in Cadmium (Cd) accumulation among rice cultivars, and concentrations in shoots and grains are generally higher in indica rice cultivars than in japonica rice cultivars, but the mechanism remains unknown. The main objective of this study was to investigate differences in heavy metal accumulation between rice subspecies through the analysis of 46 indica cultivars and 30 japonica cultivars. At the seedling stage, the mean Cd concentrations in the shoots of indica subspecies were significantly higher than those in japonica subspecies (1.22-fold), but this pattern was not observed in the roots. At the filling stage, the mean Cd concentrations in the shoots and spikes of indica subspecies were 1.66- and 2.14-fold higher than the respective concentrations in japonica subspecies. At the harvest stage, the mean Cd concentrations in the shoots and brown rice of indica subspecies were 1.61- and 2.27-fold higher than the respective concentrations in japonica subspecies. These results indicate that root-to-shoot and shoot-to-grain translocation, rather than Cd absorption in the roots, may be the key processes that determine the differences in Cd accumulation among rice subspecies. Gene expression analysis revealed that overall, the expression levels of the Cd transporter gene OsNramp1 notably increased (22.46-fold), but the expression levels of OsHMA2, OsHMA3 and OsNRAMP5 were not significantly changed at the seedling stage in the 76 cultivars exposed to Cd; the expression levels of OsNramp1 were positively correlated with the Cd concentrations in spikes at the filling stage. In addition, a significant difference was observed in the expression levels of OsNramp1 between the indica and japonica subspecies, which may explain the higher Cd concentrations in roots but lower Cd concentrations in spikes and brown rice for the japonica subspecies. Together, these results demonstrate that OsNramp1 may be the most important gene among the four selected genes in the promotion of Cd uptake by roots and transfer of Cd into spikes and eventually into brown rice.     

The role of auxins and cytokinins in the mutualistic interaction between Arabidopsis and Piriformospora indica

Arabidopsis growth and reproduction are stimulated by the endophytic fungus Piriformospora indica. The fungus produces low amounts of auxins, but the auxin levels and the expression of auxin-regulated genes are not altered in colonized roots. Also, mutants with reduced auxin levels (ilr1-1, nit1-3, tfl2, cyp79 b2b3) respond to P. indica. However, the fungus rescues the dwarf phenotype of the auxin overproducer sur1-1 by converting free auxin into conjugates, which also results in the downregulation of the auxin-induced IAA6 and the upregulation of the P. indica-induced LRR1 gene. The fungus produces relatively high levels of cytokinins, and the cytokinin levels are higher in colonized roots compared with the uncolonized controls. trans-Zeatin cytokinin biosynthesis and the CRE1/AHK2 receptor combination are crucial for P. indica-mediated growth stimulation, while mutants lacking cis-zeatin, impaired in other cytokinin receptor combinations, or containing reduced cytokinin levels respond to the fungus. Since root colonization is not affected in the cytokinin mutants, we propose that cytokinins are required for P. indica-induced growth promotion. Finally, a comparative analysis of the phytohormone mutants allows the conclusion that the response to P. indica is independent of the architecture and size of the roots.     

Characterization of cold-induced changes in the fatty acids profile of rice seedlings

Rice, a staple food for more than one half of the world’s population, is one of the most cold-sensitive cereals. Breeding programs aimed at increasing rice production are expected to reduce cold-imposed grain losses. Several reports have demonstrated that cold induce differential effects on the fatty acids profile of membranes in chilling-sensitive and chilling-tolerant plants. In this work, we evaluated changes in fatty acid (FA) composition as a potential screening tool to evaluate chilling sensitivity of rice accessions. Cold exposure led to the preferential accumulation of the polyunsaturated linolenic and linoleic FAs and reduction of palmitic and stearic FAs, besides showing increased lignoceric acid content in roots of the variety. Similarly, roots of cold-exposed line Quila 66304 also presented preferential accumulation of linolenic and linoleic FAs and reduction of palmitic and stearic FAs. Cold exposure also led to enhanced levels of palmitic acid in shoots of Amaroo and, in a smaller extent, in shoots of Quila 66304. Linolenic acid was reduced in the shoots of both Amaroo and Quila 66304, while oleic acid content was reduced in shoots of Amaroo and slightly increased in shoots of Quila 66304. Double-bond index analysis indicated that 18 carbons FAs DBI for roots might be a good screening tool for cold response in rice. Results in this report demonstrate that cold-induced changes in FA profile represent a useful screening tool for early identification of differences in cold acclimation potential among rice accessions.     

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs).     

Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses.

Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication.